Increasing cell culture density during a developmental window prevents fated rod precursors derailment toward hybrid rod-glia cells

In proliferating multipotent retinal progenitors, transcription factors dynamics set the fate of postmitotic daughter cells, but postmitotic cell fate plasticity driven by extrinsic factors remains controversial. Transcriptome analysis reveals the concurrent expression by postmitotic rod precursors of genes critical for the Müller glia cell fate, which are rarely generated from terminally-dividing progenitors as a pair with rod precursors. By combining gene expression and functional characterisation in single cultured rod precursors, we identified a time-restricted window where increasing cell culture density switches off the expression of genes critical for Müller glial cells. Intriguingly, rod precursors in low cell culture density maintain the expression of genes of rod and glial cell fate and develop a mixed rod/Muller glial cells electrophysiological fingerprint, revealing rods derailment toward a hybrid rod-glial phenotype. The notion of cell culture density as an extrinsic factor critical for preventing rod-fated cells diversion toward a hybrid cell state may explain the occurrence of hybrid rod/MG cells in the adult retina and provide a strategy to improve engraftment yield in regenerative approaches to retinal degenerative disease by stabilising the fate of grafted rod precursors.

In proliferating multipotent retinal progenitors, transcription factors dynamics set the fate of postmitotic daughter cells, but postmitotic cell fate plasticity driven by extrinsic factors remains controversial. Transcriptome analysis reveals the concurrent expression by postmitotic rod precursors of genes critical for the Müller glia cell fate, which are rarely generated from terminallydividing progenitors as a pair with rod precursors. By combining gene expression and functional characterisation in single cultured rod precursors, we identified a time-restricted window where increasing cell culture density switches off the expression of genes critical for Müller glial cells. Intriguingly, rod precursors in low cell culture density maintain the expression of genes of rod and glial cell fate and develop a mixed rod/Muller glial cells electrophysiological fingerprint, revealing rods derailment toward a hybrid rod-glial phenotype. The notion of cell culture density as an extrinsic factor critical for preventing rod-fated cells diversion toward a hybrid cell state may explain the occurrence of hybrid rod/MG cells in the adult retina and provide a strategy to improve engraftment yield in regenerative approaches to retinal degenerative disease by stabilising the fate of grafted rod precursors.
The vertebrate retina extracts the most relevant features from the visual scene for further processing by higher visual structures 1 . Retina operation relies on three-dimensional neural networks, neatly organised in layers populated by neurons with sharply different morphologies and functional properties 2,3 . In the retina, neurons and glial cells stem from a common progenitor 4,5 , whose developmental potential progressively restricts to generate stage-specific intrinsically different progenitors, which give birth to a limited repertoire of retinal cell types (recently reviewed in [6][7][8]. Transcription factors (TFs) in progenitor cells provide the intrinsic cues setting the fate of their postmitotic daughter cells, and additional specification steps may play a role in stabilising the fate of immature postmitotic precursors. Indeed, extrinsic factors such as cell culture density may promote a fate reassignment, although limited to the cell types generated by a given precursor at a given developmental time [9][10][11] . For instance, a reduced number of rod precursors may increase amacrine or bipolar cells, but rod precursors may not convert to earlyborn cone precursors.
At variance with this notion, late-born rod precursors lacking the postmitotic specification signals provided by TFs such as Neural Retina Leucine Zipper (Nrl) 12,13 or Nuclear Receptor Subfamily 2 Group E Member 3 (Nr2e3) 14,15 may undergo a fate diversion toward early-born blue cones 16 . It is presently unclear whether the An increase in cell culture density selectively downregulates the expression by rod precursors of MG genes. Diffusible signals generated by retinal neurons may operate as extrinsic factors controlling rod precursors fate stabilisation [9][10][11] as a function of cell culture density.
A recent analysis of retinal stiffness indicates a gradient across layers, increasing with the number of cells for unit volume from the ganglion cells layer to the ONL in both ruminants 37 and mouse retinas 38 . Accordingly, ONL stiffness is expected to increase during development, initially due to the increase in rod cell number, then The Y-axis plots the rate of rod precursor generation in arbitrary units. Precursors appear around embryonic day 13 (E13), the rate of generation peaks around the day of birth (PN0), and the process ends around postnatal day 6 (PN6). The arrowhead indicates the time of transcriptional shift in rod precursors. Arrows at PN4 and PN8 indicate the time of GFP + rod precursors sorting for the evaluation of transcriptional changes by RNAseq analysis. (b) Scatter plot of log 2 Transcripts per Million reads (TPM) at PN8 vs Log 2 (TPM) at PN4. Each gene is plotted with X and Y coordinates representing normalized expression at PN4 and PN8, respectively. (c) Heatmap representing statistically significant GO over-represented classes associated with differentially expressed genes (fold change > |1.5| and false discovery rate = 0.05). www.nature.com/scientificreports/ To assess the role played on rod vs MG fate stabilization by factors linked to the number of cells for surface area/volume, we disrupted cell-cell contacts by enzymatic treatment followed by mechanical dissociation and plated isolated retinal cells at either low (1 ×-9.4 × 10 4 cells/cm 2 ) or high (4×-3.77 × 10 5 cells/cm 2 ) cell densities (see also "Materials and methods"). As shown in Fig. 3a, we selected the day of birth (PN0) for cell isolation to ensure enough cells for subsequent analysis and a timespan to adapt to the in vitro conditions and develop a response to cell culture density. As a preliminary step, we evaluated whether isolated rod precursors maintain their electrophysiological identity in culture by monitoring the Cs-sensitive hyperpolarisation-activated current (I HYP ), a prominent inward current of adult rods expressed by both mice 39 and human rod precursors 40 . A substantial fraction of I HYP flows through ion channels coded by Hcn1, a gene already expressed at an early developmental time and whose expression progressively builds up till PN28 (adult). Although Hcn1 is not a rod-specific gene and is not among the genes most upregulated between PN4 and PN8, it is expressed by most primary sensory neurons 26 , and I HYP measurement may indicate whether rod precursors maintain their sensory neuron phenotype in culture. Furthermore, recent evidence in human rod precursors of iPSC-derived retinal organoids indicates that the current carried by HCN1 channels is sensitive to 4-hydroxy tamoxifen 40 , a blocker of estrogen-related receptor beta (ERRβ) critical for rod viability 41 coded by the DEG Esrrb.
Surprisingly, 5 out of 19 PN0/DIV8 GFP + cells lacked I HYP and had an electrophysiological signature akin to MG ( Supplementary Fig. S1). This unexpected observation may indicate that some rod-fated precursors in 1 × cell density cultures deviate from their initial fate assignment, drifting toward the MG fate, a notion consistent with the hybrid transcriptional profile of sorted GFP + rod precursors, which express c-Kit and Mcam along rod-specific genes up to PN4.
A critical issue with transcriptomic data from pooled rod precursors generated across a broad developmental window 4,42 is that they may provide a blurred picture of the intrinsic dynamics of single rod precursors. For instance, there is no evidence that a specific c-Kit-expressing cell in a pooled cell population would also express the rod-specific genes Rho or Mcam, a gene critical for MG development 36 . Furthermore, rod precursors expressing GFP in response to Nrl may eventually deviate from their initial fate assignment and start expressing genes of different fates while maintaining the GFP fluorescence due to the high protein stability.
To circumvent these possible issues, we measured the expression of down-regulated genes such as c-Kit, Mcam, and Tnfsf9 along with the rod-specific gene Rho and Hcn1 in single GFP + cells functionally characterised by patch-clamp recording and cultured at either 1 × or 4 × cell densities. As shown in Fig. 4a, assuming a similar developmental time in vivo and in vitro, we collected PN0/DIV2, PN0/DIV4 and PN0/DIV8 GFP + rod precursors for single-cell qRT-PCR. As shown by data in Fig. 4, both c-Kit (Fig. 4b) and Mcam (Fig. 4c) expression remained high in PN0/8DIV GFP + cells cultured at the 1 × cell culture density (open circles), but their expression decreased significantly (***P < 0.001, see figure legend) in cells cultured at the 4 × cell density (filled circles).
The influence of cell culture density appears specific, with no effect on − ΔC t of Tnfsf9 (Fig. 4d), another gene down-regulated during rod specification. Cell density affected neither Rho (Fig. 4e) nor the housekeeping gene Actb (Fig. 4g) expression, but Rho expression significantly increased with time in culture (*P < 0.05, for the comparison between PN0/DIV2 and PN0/DIV8, see legend) independently of cell culture density.
Consistent with single-cell expression data, immunostaining showed c-Kit labelling of several PN0/DIV8 cells cultured at the 1 × cell culture density, including the GFP + cell (yellow arrowheads in Fig. 4h,i), plotted at higher magnification in the inset. On the other hand, PN0/DIV8 cells cultured at the 4 × cell culture density did not exhibit c-Kit labelling, either by GFP + or GFP − cells (Fig. 4j). In agreement with single-cell data, cell culture density does not affect Rho labelling in PN0/DIV8 cells.
Consistent with data in Fig. 3h indicating a non-significant increase in G HYP in PN0/DIV8 cells, Hcn1 expression did not significantly increase with time in culture (Fig. 4f). However, the observation that PN0/DIV8 GFP + cells with an electrophysiological profile of MG-like cells, i.e., lacking I HYP ( Supplementary Fig. S1), had − ΔC t values for Hcn1 (open diamonds) similar to rod-like cells (circles) (Fig. 4e), suggests a mismatch between Hcn1 expression and normalised Cs-sensitive G HYP . We investigated this mismatch by plotting in Fig. 5 normalised G HYP as a function of − ΔC t values separately for PN0/DIV4 and PN0/DIV8 rod precursors cultured at 1 × cell density. G HYP values of PN0/DIV4 cells cultured at the 1 × density (Fig. 5a, open circles) showed a moderate increase with Hcn1 − ΔC t values spanning from − 7.2 to − 0.5, i.e. an over 2-log 10 units change in Hcn1 expression.
We evaluated the presence of a second Cs-sensitive inward rectifying current (I ir ) as a possible cause of the inverse relationship between G HYP and Hcn1 expression in a fraction of GFP + cells. This notion is intriguing because Kcnj10, which codes for inward rectifying potassium channels (K ir 4.1), appears not dispensable for the MG cell fate, and its expression progressively increases during MG postnatal development 36 . Figure 5 shows the impact of Ba 2+ , a K ir channels blocker that does not affect HCN1 channels (I h ), on I HYP of PN0/DIV8 cells cultured at the 1 × cell density ( Fig. 5c-h). Ba 2+ action ranges from a lack of effect ( Fig. 5c-e) to an over 50% block of I HYP (Fig. 5f,g), with the residual I h abolished by Cs (Fig. 5h).
In 6 rod precursors, Ba 2+ blocked a variable fraction of I HYP (Fig. 5k), suggesting that PN0/DIV8 cells cultured at 1 × cell density (Fig. 5b)    www.nature.com/scientificreports/ GFP + cell cultured at the 4 × density (Fig. 5b, filled circle) had a lower G HYP than cells with similar Hcn1 − ΔC t but cultured at a lower cell density.
Cell culture density affects rod precursors maturation over a limited time window. Some PN0/ DIV8 GFP + cells cultured at 1 × cell density may escape fate derangement toward a hybrid phenotype, as suggested by their low G HYP with high Hcn1 expression ( Fig. 5b) or by the lack of Ba 2+ -sensitive I HYP (Fig. 5c-e). GFP + cells escaping diversion to the hybrid phenotype may result from cell culture density impacting rod precursors during a restricted time window. Considering that increased cell culture density strongly suppressed c-Kit and Mcam expression in PN0/DIV4 rod precursors, we investigated whether cell density would impact rod precursors cultured 4-day in vitro following isolation at PN4 (PN4/DIV4), i.e., after they had proceeded up to PN4 through the specification steps in their native retinal environment rather than in culture (Fig. 6a), but before the occurrence of the transcriptional switch at PN6 24 . Figure 6 shows I HYP recorded from two PN4/DIV4 GFP + cells cultured at 4 × (Fig. 6b) and 1 × (Fig. 6c) cell densities and, for comparison, the PN0/DIV8 GFP + cell from Fig. 3 cultured at the 1 × cell density (Fig. 6d). Analysis of activation parameters indicated a trend toward G HYP reduction in PN4/DIV4 cells (Fig. 6e). In addition, the half-activation voltage (V 0.5HYP ) of PN4/DIV4 cells cultured at the 4 × cell density (filled circles) (Fig. 6g) shows a borderline shift (see legend) toward less negative V 0.5 . According to data in Fig. 5, the occurrence of GFP + precursors downregulating Hcn1 expression while shifting toward MG-like cells by upregulating current through Ba 2+ -sensitive Kir channels may increase G HYP variability in PN0/DIV8 compared to PN4/DIV4 cells. The observation of similar variability in the normalized conductance of PN4/DIV4 cells at the two culture densities suggests that most rod precursors at PN4 have lost their sensitivity to cell culture density.
This notion is consistent with gene expression analysis comparing PN4/DIV4 to PN0/DIV8 cells ( Fig. 6h-k). At variance with data collected in PN0/DIV4 rod precursors in Fig. 4, c-Kit (Fig. 6h) and Mcam (Fig. 6i) expression in PN4/DIV4 cells did not change with culture cell densities. However, PN4/DIV4 cells (circles) show a significantly reduced (*P < 0.05, see legend) Mcam expression compared to PN0/DIV8 cells cultured at 1 × cell density (squares, Fig. 6i). On the other hand, cells isolated at PN4/DIV4 cells (circles) had − ΔC t values for c-Kit (Fig. 6h) not significantly different from those of PN0/DIV8 cells (squares) cultured at 1 × cell density. It is relevant to note that even for Mcam, the reduction in its expression in PN4/DIV4 cells is far less pronounced than that observed in PN0/DIV4 cells for changes in cell density. Overall, these observations suggest a substantially decreased sensitivity to changes in culture cell density by cells isolated at PN4 compared to those isolated at PN0.
In PN4/DIV4 cells (circles), both Rho (Fig. 6j) and Hcn1 (Fig. 6k) − ΔC t values were similar at either culture densities and not significantly different from those of PN0/DIV8 GFP + cells cultured at low cell density (squares). Data in Fig. 5, showing the increase in Ba 2+ -sensitive G HYP in PN0/DIV8 cells in the 1 × cell culture density, suggest that the low cell culture density may not simply fail to switch off the expression of genes critical for MG cells development, but it may promote the development of MG functional properties. To provide additional evidence for this point, we exploited the response of MG to inflammatory stimuli, such as bacterial lipopolysaccharide (LPS), which induced the release of inflammatory cytokines in the medium by both human and mouse MG 43,44 . As shown in Fig. 7, LPS treatment induced a significant increase of inflammatory cytokines IL-6 ( Fig. 7a) and TNF-α (Fig. 7b) in the culture medium of PN0/DIV8 compared to untreated controls. Intriguingly, LPS treatment did not trigger cytokines release in the medium by PN0/DIV4 cells, consistent with the notion that 1 × density culture provides permissive conditions for developing MG properties after DIV4.

Discussion
The notions of fate reassignment in postmitotic cells and extrinsic factors' roles in promoting fate diversion represent unsettled issues.
Transcriptional data in Fig. 1 and Table S1 from pooled rod precursors indicated that GFP + rod precursors (sorted from retinas of Nrl-GFP + transgenic mice) express genes critical for MG development (Mcam) and also genes common to retinal progenitors and MG (c-Kit) up to PN4. Furthermore, molecular analysis by qRT-PCR in single retinal cells (Fig. 4) indicated the simultaneous expression by GFP + PN0/DIV4 of a retinal progenitor gene (c-Kit), as well as of a gene critical for MG cells development (Mcam) along with rod-specific (Rho) and primary sensory neurons-expressed genes (Hcn1). Overall, these findings support the notion of intrinsically hybrid rod precursors and oppose the possibility that transcriptome data in Fig. 1 and Supplementary Table S1 may result from the pooling of GFP + cells at different stages of maturation rather than their intrinsic hybrid features. Furthermore, immunostaining for c-Kit in ONL GFP + cells at PN4 (Fig. 2) and in cultured PN0/DIV4 cells (Fig. 4) suggest ongoing c-Kit transcription by GFP + rod precursors, i.e., by bonafide rod-fated cells.
The hybrid transcriptional profile of rod precursors up to PN4, i.e., well after the peak of rod generation at PN0, suggests fate stabilisation does not occur stepwise, providing a window for rod precursors fate reassignment during their early postmitotic development.
We explored cell culture density's role in either fate stabilisation or reassignment by assessing its ability to suppress genes common to MG and late progenitor cell fates and observed a highly-significant suppression by PN0/DIV4 rod precursors of both c-Kit and Mcam in response to a fourfold increase in cell culture density (Fig. 4). Moreover, cell culture density affected neither the rod-specific gene Rho nor Actb, suggesting a specific action on downregulated genes of different fates.
The observation that an increase in cell culture density does not suppress the expression of Tnfsf9, a gene downregulated during photoreceptor development, may indicate that multiple extrinsic factors control rod precursors' postnatal maturation. Indeed, in the developing mouse retina, Hypoxia-inducible factor-alpha (coded by Hif1a) stabilisation by the propyl isomerase inhibitor Roxadustat 45  Cell culture density has previously been reported as an extrinsic factor able to redirect rod precursors toward a different late-born neuronal fate 9 . In agreement with the notion of cell density operating within a restricted time window, we found that changes in culture cell density did not significantly affect c-Kit and Mcam expression by retinal cells isolated at PN4 and cultured 4 days in-vitro (PN4/DIV4), suggesting that rod precursors sensitivity to cell culture density subsides after PN4. In PN4/DIV4 rod precursors, Mcam levels fell between those of PN0/ DIV4 cells cultured at two different cell densities. This observation may indicate rod precursors maturing in the native retinal environment had already started suppressing Mcam expression by PN4, but without reaching the extent observed in the 2 × culture conditions, in agreement with data in Supplementary Table S1 and Fig. 2 showing c-Kit expression and immunostaining in the retina up to PN4. The observation of similar c-Kit expression levels in PN4/DIV4 and PN0/DIV8 rod precursors cultured at the 1 × cell density may indicate a different sensitivity to cell culture density between Mcam and c-Kit.
The effects of cell culture density on rod precursors maturation have been attributed to low molecular weight diffusible factors 9,46 . Taurine 47 and retinoic acid 48 have been reported to promote rod precursors maturation or steer retinal progenitors toward the rod fate at the expense of other late retinal-born neurons (reviewed by 7 ). However, the impact of cell density in our cultures does not appear to recapitulate the effects of known small molecular weight diffusible factors on rod precursors 9 . First, cell density effects occur irrespectively of cells being exposed to the same retinoic acid level (1 µM in the culture media). In addition, patch-clamp recordings from GFP + cells in Fig. 3 indicate they display functional properties akin to rod precursors rather than bipolar cells, as previously reported for cells cultured in 1 × density 9,46-48 .
Cell-cell contacts and tissue stiffness are two crucial differences between our experimental conditions and those used by Altshuler and Cepko 9 , which embedded dispersed retinal cells in a three-dimensional collagen matrix to avoid cell-cell contacts and provide a soft matrix for cells 9 . On the other hand, we cultured cells as a two-dimensional system, allowing cells to establish cell-cell contacts. Indeed, recent evidence indicates that a stiffness gradient occurs across retinal layers linked to the number of cells per unit volume 37,38 , suggesting that ONL stiffness may progressively increase during development, mirroring the increase in ONL cell number.
We did not investigate in detail the molecular mechanisms underlying cell culture density impact on rod precursors' maturation. However, the expression of transcription factor Yes-associated protein (Yap1), known for its role in mechanotransduction 49,50 , was not detectable in PN4/DIV4 GFP + cells, a finding consistent with the downregulated Yap1 expression found between PN4 and PN10 in rod precursors transcriptomic data 24 . Yap1 is expressed in adult MG cells 51 , where the Hippo pathway inhibits its nuclear translocation 50,52 to prevent MG cells proliferation, but it is presently unknown whether Yap signalling in rod precursors may depend on Mcam, as recently reported for glioblastoma cells 53 . Although comparing the transcriptome of sorted PN0/DIV4 GFP + rod precursors cultured using different cell densities may hint at the underlying molecular events, single-cell transcriptome analysis may avoid the shortcomings of substantial cell loss when sorting cultured GFP + rod precursors.
An intriguing point is whether 1 × cell culture density may only prevent rod precursors from switching off the expression of immature genes, such as Mcam and c-Kit, or it may promote the MG fate. Data in Fig. 7 indicate that low cell culture density provides permissive conditions for developing MG functional properties in PN0/DIV8 retinal cells, although they do not prove that may happen in rod precursors. However, the analysis of P0/DIV8 rod precursors cultured at 1 × cell density revealed that 5 out of 19 GFP + cells developed functional properties akin to Muller glial cells, as judged by the lack of I HYP in response to membrane hyperpolarisation, while Rho, Hcn1, and Tnfsf9, expression levels remained similar to GFP + cells displaying typical rod precursors functional features. Interestingly, the lack of I HYP despite HCN1 expression has previously been reported for rod precursors derived from adult human MG cells 54 , and mouse ciliary margin-derived Nrl-expressing cells also lack Cs-sensitive I HYP despite Hcn1 expression 55 . These findings suggest that low cell culture density may not just   www.nature.com/scientificreports/ These molecular and functional data suggest that some PN0/DIV8 GFP + cells cultured at the 1 × density may progressively derail from the rod fate toward a hybrid cell type with rod precursor features coexisting with MG cell properties.
The notion of cells with a hybrid cell profile has already been proposed for rod precursors of rd7 mice lacking the TF Nr2e3 17 . Since rod precursors and MG usually do not originate from the same terminally-dividing late progenitor 25 , the finding of rod precursors failing to interconvert with MG cells fits the notion that a fate reassignment may only occur between cell pairs originating from terminally-dividing progenitors. Intriguingly, recent evidence based on single-cell transcriptomic in cells isolated from the adult mouse retina indicates the occurrence of a cell cluster with a hybrid photoreceptor-MG feature and the expansion of this cluster in response to retinal degeneration 60 .
The evidence that cell culture density is an extrinsic factor affecting rod precursors maturation may extend beyond developmental biology, impacting the regenerative approach to treating retinal dystrophies. Transplanting either unsorted retinal cells (4 × 10 5 cells µl −1 ) or donor mouse NRl-GFP rod precursors (2 × 10 5 cells/µl −1 ) isolated at PN1 leads to similar integration rate into the host retina 61 . However, photoreceptor replacement via photoreceptor precursors transplantation has been challenged by the occurrence of material transfer between donor and host cells via a cytoplasmic exchange rather than by replacement [62][63][64] in an environment-dependent manner 65 . Intriguingly, in a model of terminal photoreceptor degeneration, a significant improvement in the yield of truly integrating donor cells has been reported upon increasing the number of sorted transplanted cone precursors for unit volume 66 from 2 × 10 5 cells µl −1 to 3.33 × 10 5 cells µl −1 . For our cultures on a 13 mm round coverslip, assuming cells may pile up to 5 µm height, culture cell density would range from about 7.5 × 10 5 cells µl −1 (4 ×) to 1.9 × 10 5 cells µl −1 (1 ×). These figures suggest that the improvement in transplantation outcome occurs within the range of cell densities affecting the downregulation of immaturity genes in rod precursors. Evaluation of grafted cells may assess the possible derailment of grafted rod precursors toward a hybrid phenotype upon transfer to the subretinal space, i.e. to a location whose stiffness and pO 2 levels (recently reviewed in 67 ) may differ from the ONL, where rod maturation usually occurs.

Materials and methods
Animals. To improve breeding efficiency, heterozygous NRL:GFP + transgenic mice (kindly provided to Prof. Vania Broccoli by Prof. A. Swaroop) were bred with Wild Type CD1 (The Jackson Laboratory, Bar Harbor, ME). Animals husbandry and retina isolation did comply with the guidelines for animal research of the Association for Research in Vision and Ophthalmology, the European Community and Italian laws. The protocol, complying with ARRIVE guidelines, was approved by the Italian Ministry of Health ethical committee (authorization N° 45/2016 of January 16, 2016). The animals were genotyped by ear biopsy using the following PCR primers: NrlGFP-geno-Fw: 5′CTG AAT ACA GGG ACG ACA CCAGC3′. NrlGFP-geno-Rv: 5′CGT AGG TCA GGG TGG TCA CGAG3′.
Isolation and dissociation of mouse retinas. Eyes enucleated at PN0 or PN4 after cervical dislocation under isoflurane anaesthesia, and isolated retinas were washed in HBSS with 0.1% Gentamicin and 1% Penicillin/Streptomycin. Digestion in HBSS containing 10 U/ml Papain (Worthington Biochemical Corporation, USA), 0.5 mM EDTA, and 1.5 mM cysteine for 12 min at 37 °C, was followed by mechanical dissociation via passages through a 1000 µl micropipette tip. After centrifugation for 10 min at 180×g, cells were resuspended in DMEM:F12 medium with 2 mM of l-Glutamine, 1% Penicillin/Streptomycin, 1% N2 supplement, 2% B27 supplement and 0.5 μM Retinoic Acid. Cells were counted by the Trypan blue exclusion method with a Burker Chamber and seeded on 13 mm glass coverslips previously coated with Poly-d-Lysine and 1% Matrigel at two different cell densities: 0.5 × 10 6 and 2 × 10 6 cells were spread over the 1.33 cm 2 glass coverslip, resulting in cell culture densities of 3.77 × 10 5 cells/cm 2 (1 × density) and 1.51 × 10 6 cells/cm 2 (4 × density) for coverslip, respectively. Cells were routinely cultured for 2-4-6-8 days in vitro (DIV) at 37 °C in a humidified atmosphere of 5% CO 2 , and the medium changed every other day.

Transcriptome analysis of NRL-GFP cells. Using the GFP signal, NRL-GFP cells from PN4 and PN8
transgenic mice were sorted by a Fluorescence-Activated Cell Sorting (FACS) system. For every time point, the quality and concentration of total RNA extracted from three sorted biological replicas using the RNeasy mini kit (Qiagen) were assessed with NanoDrop (Thermo Scientific). RNA-seq libraries preparation and sequencing were outsourced to IGATech (Udine, Italy). Libraries were generated according to the TruSeq Stranded Total RNA Ribo-Zero Gold protocol (Illumina, San Diego, CA) and sequenced using the Illumina HiSeq2500 platform with a 125-bp paired-ends design. Three biological replicates were generated for each group of transgenic mice. On average, for each sample, we generated more than 30 million paired-end reads with a quality score (Phred-score) > 30. Raw reads were processed to filter out low-quality reads and adaptors using the program Cutadapt and the wrapper tool TrimGalore v0.6.2 (http:// www. bioin forma tics. babra ham. ac. uk/ proje cts/ trim_ galore/) with default parameters. Clean reads were used to quantify the annotated Mus musculus gene models, assembly version 38.87 (http:// www. ensem bl. org), with the program Salmon 68 .
Briefly, clean reads were mapped to the reference transcriptome using Salmon quant command with default parameters except for the options --numBootstraps 30 Immunofluorescence on retina slices and Immunocytochemistry. Immunofluorescence on retina slices was performed as previously described 71 . In particular, whole eyes were enucleated at PN4 and PN8, immediately fixed in 4% Paraformaldehyde for 2 h and incubated overnight (O/N) in 15% w/v sucrose solution. The next day, eyes were transferred in cryomolds, submerged with Tissue-Tek ® O.C.T. Compound, and sectioned using a cryostat. For immunofluorescence staining, glass slides were reheated 20 min at RT, blocked in 10% BSA in PBS 1 × for 2 h at RT, and incubated with the primary antibody at 4 °C. After incubation with the secondary antibody, the sections were mounted with VECTASHIELD ® Antifade Mounting Medium with DAPI.
Secondary antibodies: Alexa Fluor 568 donkey anti-rabbit IgG, 1:500 (Molecular Probes, Eugene, OR, USA). All images were acquired on a Nikon Ti-e microscope with an A1 scanning head and 405/488/561/630 laser lines, using a 40 × water immersion objective (Nikon APO NA 1.25) for tissue imaging and 100 × oil immersion objective (Nikon APO TIRF NA 1.49). Zoom and image dimensions were adjusted to obtain a pixel size of 80 nm, and the pinhole was set at Airy 1. A 2 × 2 binning was performed on the acquired images. Laser power was maintained below 0.5% to minimize photobleaching.
Electrophysiological recordings from cultured rod precursors. A coverslip was transferred to a recording chamber bottomed by a 0.1 mm-thick coverslip mounted on the stage of an inverted microscope equipped with a 63 × objective, a cooled CCD camera, and GFP fluorescence excitation/emission filters. A manifold with electrovalves controlled the switch between saline solutions used to perfuse cells during recording. GFP + rod precursors cells were approached by the patch-pipette using a motorised micromanipulator. Brief treatment with 0.5 mg/ml hyaluronidase increased the success rate of gigaseal formation with cells cultured at 4 × cell density.
We used the perforated-patch technique to prevent I h rundown during whole-cell recording, as previously reported for adult mouse rods [72][73][74] and ciliary margin-derived cells 55 . Patch pipettes were drawn from 1.5 mm OD borosilicate glass (Hirschmann) by an air-cooled two-stage horizontal puller, with series resistances ranging from 50 to 80 MΩ, which proved satisfactory for cells with resistances higher than 3 GΩ, membrane capacitance 3-7 pF, and currents with activation time constants longer than 50 ms. When collecting samples for single-cell qRT-PCR, we controlled the filling level of the pipette with the pseudo-intracellular solution to minimise either dilution or concentration of mRNA.
Voltage-gated currents were filtered at 300 Hz by an EPC-8 amplifier, and currents acquired at 1 kHz sampling rate using a 16-bit A/D board were stored in the computer hard disk for later analysis by Origin 8.5.1 Pro (Microcal).
Recordings started in Locke's solution, an isotonic saline solution allowing both adult and immature mouse rods survival for several hours 55,74 . High K saline used to amplify I HYP contained (in mM): NaCl, 120; KCl, 25; CaCl 2 , 2, MgCl 2 , 2.4; glucose 10; Hepes, 10-pH 7.4. I HYP block was induced by a high K solution with either CsCl or BaCl 2 to final concentrations of 3 or 2 mM. Patch-pipetted were filled with a pseudo-intracellular solution containing (in mM): NaCl, 10; KCl, 140; Pipes, 10-pH 7.2 with KOH. All salts were from Sigma-Aldrich, Italy.

Analysis of cytokine secretion by dissociated cells after stimulation with LPS. Cells from PN0
retinas were cultured at 1 × cell density and treated at DIV3 or DIV7 for 24 h with 0.1 μg/ml of lipopolysac- www.nature.com/scientificreports/ charide (LPS). At the end of treatment, 200 μl of culture media were collected, and pro-inflammatory cytokines IL-6 and TNF-α measured by ELISA assay performed according to the manufacturer's instructions (TNF alpha Mouse ELISA Kit and IL-6 alpha Mouse ELISA KIT, Thermo Fisher Scientific, USA) and expressed as ng/ml. The experiment was performed in biological triplicate.

Statistical analysis.
Except for gene over-representation in GO (see "Transcriptome analysis of NRL-GFP cells" above), data analysis was carried out using Origin 8.5.1. The normality of data distribution was tested by the Shapiro-Wilk test. The impact of time in culture (DIV) and treatment (cell culture density) on I HYP and single-cell expression were analysed by two-way ANOVA. In addition, 1-way ANOVA was used to analyse cytokine secretion in the medium and compare membrane capacitance (Cm) between adult MG cells and PN0/ DIV8 cells in Supplementary Fig. S1. Multiple comparisons were carried out using Bonferroni's correction for a two-tailed t test.